This proposal involves an investigation of the enzyme chitin synthetase from the crustacean Artemia salina and a comparison of its properties with published data on the analogous enzyme from fungi, e.g. Saccharomyces cerevisiae. Also, the effects of inhibitors such as difluobenzuron and polyoxin D on the crustacean enzyme will be examined in order to differentiate and characterize the two chitin secreting systems. A model system for this research will be larvae of the brine shrimp, Artemia salina and the blue crab, Callinectes sapidus. Microsomes will be obtained by nitrogen bomb cavitation of larvae, and differential centrifugation; if needed, plasma membranes will be prepared by sucrose density gradient centrifugation. Crustacean chitin synthetase will be assayed as described by Carey (1965). Incubations will be extracted with (a) 50% methanol, (b) chloroform:methanol (2:1), (c) chloroform:methanol:water (10:10:3) and (d) 9.5 M urea. Approximately 66% of the radiolabeled product has been identified as chitin by chemical and enzymatic methods. The proposed research will include an examination of the role of oligosaccharide- lipids in the biosynthesis of crustacean chitin, the glycosylation of endogenous and exogenous primers by chitin synthetase and the possible inhibition of these processes by DIMILIN and Polyoxin D. Finally, the linkage region between crustacean chitin and protein will be examined by chemical and proteolytic treatment of radiolabeled product followed by purification radioactive chitopeptides and amino acid analysis. These studies are expected to provide further evidence that crustacean chitin is secreted as a protein-polysaccharide complex and not as a pure homopolysaccharide. Results obtained using the crustacean enzyme will be compared with data on the fungal enzyme in order to more clearly understand the latter enzyme as a possible target for fungicidal antibiotics. The effect of a potentially harmful pesticide, DIMILIN, on the biosynthesis of crustacean chitin will be studied at the subcellular level. Such studies are essential before this drug is released to the general market.